1. Field of the Invention
This invention relates to a novel organosilicon Si—O—R type of compound which is a multiple fluorescent natural dye purified from the body wall extract of a marine invertebrate Holothuria scabra belonging to the Phylum: Echinodermata, Class: Holothuroidea, Order: Aspidochirota, Family: Holothuroidae.
2. Background of the Technology
The compound is a polysacchride fluorochrome having a phenolic fluorophore part and is connected to a silicon matrix around it through sulphate bonds. This silicon part is an integral part of the core molecule and takes part in the metabolism of the animal. The compound is rich in sulphur. The invention also provides a process for the extraction, purification and characterization of the novel compound and the multiple fluorescent dye from a living marine organism, especially sea cucumber. The invention also discloses for the first time chemical structure of a novel fluorescent compound where silicon has become the integral part of the organic molecule and that of its phenolic type of its fluorophore. It further provides the unusual properties of the compound and characteristics of the dye and discloses their advantages. It also describes industrial utilities of the novel fluorescent compound as a multiple fluorescent natural dye for non-radioactive labeling for florescent in situ hybridization, Flow cytometry, Immunoassays and fluorescence microscopy. The compound has another utility for companies dealing in marketing of fluorescent molecular probes by providing them with a fluorescent compound of high molecular weight which has been quite sought after for some special requirements and was not available and also providing them with an alternative of low molecular weight fluorescent dyes by utilization of only its fluorophore part. Several derivatives dyes of high and low molecular weight and desirable properties for fluorescent probes for single and multiple color applications can be made. Further, the compound can be an easily miscible ingredient in compositions of cosmetic industry particularly for sun screening and as a drug in insecticidal and veterinary remedies.
Silcon is described to play an active role in the development of plants and animals. According to Lanning, F. C. (The encyclopaedia of the chemical elements edited by C. A. Hampel, Reinhold Book Corporation as subsidiary of Chapman-Reinhold.Inc, New York Page 647,1968) silicon may have played an important and necessary role, in the origin of life on the earth. Lower forms of life, such as Diatoms often use silica in their skeletal structure. He has proposed that Si—O—R type of bonds may occur in the living organisms. However, so far there is no such report available in the earlier literature where silica might have become an integral part of the organic moiety and acted as a matrix to make the compound fluorescent.
Wannagat described that the silicon content of living organisms decreases as the complexity of the organisms rises. The ratio of silicon to carbon is 250:1 in the earth's crust, 15:1 in humus soil, 1:1 in plankton, 1:100 in ferns and 1:5000 in mammals.
The same author reported that silicon plays a key, but not fully understood role in the growth of hair, nails, bones and feathers. At the site of a bone fracture silicon content increases 50-fold in the collagen web.
Organosilicon types of compounds in the market are mostly synthetic. Silicon is the generic description for an entirely synthetic polymer containing a repeating Si—O backbone. The organic groups attached to the silicon atom via silicon-carbon bonds define a class of silicone. They are sold in the market as fluids, emulsions, compounds, lubricants, resins, and elastomers or rubbers and are also used in cosmetic plastic surgery.
However, there is no information available in the literature on the silicon compound in living organisms where silica may be an integral part of the organic molecule and also have characteristics of a single or multiple fluorescent dye. Most of the currently available dyes in the market are synthetic. Stainfile-Dyes A has given a Dye index of 264 dyes. Out of which 258 are synthetic and only six are natural dyes. Production of synthetic dyes often require use of strong acids, alkalies and heavy metals as catalysts at high temperatures. This makes the processes and the effluents to be discharged an issue of environment degradation. The dyestuff industry is continuously looking for cheaper and more environmentally friendly routes to existing dyes. (Hobson and Wales, 1998. Green Dyes, Journal of the Society of Dyers and colorists (JSDC), 1998,114,42-44).
All of the available dyes are not fluorescent. Bitplane products have displayed a list of the 123 fluorochromes in the market and their excitation and emission spectrum.
(http://www.bitplane.ch/public/support/standard/fluorochrome.htm).
Fluorescent dyes are widely used in labeling of molecular probes for localizing biological structures by fluorescence microscopy e.g. in immunoassays, labeling nucleotides and oligonucleotides for in situ hybridization studies, binding to polymeric microspheres and staining of cells for use in imaging studies. Dyes are also used for selective destruction of cells such as in the technique of photodynamic therapy. (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339 issued on Sep. 27, 1988 ; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782 issued on Sep. 28, 1993).
Fluorescence is a phenomenon in which an atom or molecule emits radiation in the course of its transition from a higher to a lower electronic state. It follows stoke's law according to which the wavelength of the fluorescent radiation is always longer than that of the excitation radiation. The process of fluorescence is quite different from the phosphorescence and bioluminescence. The term fluorescence is used when the interval between the act of excitation and emission of radiation is very small (10−8-10−3 second). In phosphorescence the time interval between absorption and emission may vary from 10−3 second to several hours (R. Norman Jones, 1966 in: The encyclopaedia of chemistry, 2nd edition, 1966, Pages 435-436). Bioluminescence is the term used for the light produced as a result of a chemical reaction occurred at a particular time in a particular cell within the body of a living organism.
A large number of fluorescent dyes are reported in the Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the united States of America, 1996. In the same book on pages 1-6, Ian D. Johnson (1996) described in details the process of fluorescence & its methods of detection in certain molecules called fluorophores or fluorescent dyes by him (generally polyaromatic hydrocarbons or heterocycles). The most versatile currently in use fluorescent dyes are Fluorescein and fluorescein based and BODIPY dyes and their derivatives.
The authors have dealt in with the shortcomings of all these dyes and described their preferences of characteristics of dyes. Many derivatives of the fluorescent dyes and their synthesis are disclosed in U.S. patents (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339, issued on Sep. 27, 1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782 of Sep. 28, 1993; Kang, H. C. and Haughland, R. P, U.S. Pat. No. 5,187,288 published on Feb. 16, 1993; Kang, H. C. and Haughland, R. P in U.S. Pat. No. 5,274,113 of Dec. 28, 1993 and; Kang, H. C. and Haughland, R. P, U.S. Pat. No. 5,433,896 Jul. 18, 1995; Kang, H. C. and Haughland, R. P. U.S. Pat. No. 5,451,663 published on Sep. 19, 1995). Rosenblum Barnett B, Spurgeon S, Lee Linda G, Benson Scott C and Graham Ronald J in international patent No. WO0058406, publication date 5th Oct. 2000 reported 4,7-Dichlororhodamine dyes useful as molecular probes.
R. Norman Jones in The encycopaedia of chemistry, 2nd edition, 1966, Pages 435-436 has described specialty of a good fluorophore. According to him A fluorescent molecule must have a good chromophoric system for absorption of excitation energy and a shielding mechanism to save too rapid dissipation of the excitation energy into vibrational motion before the fluorescence retardation act can occur. He also commented that though the relationship of the molecular structure and the fluorescence of compounds are not well understood, there are certain groups, presence of which is associated with fluorescence. For example, in the organic molecules presence of phthalein and aromatic structures such as anthracene and naphthacene are particularly associated with bright fluorescence. Few inorganic compounds fluoresce strongly in the liquid state and in solids, fluorescence is often modified by the presence of trace impurities.
The inventors of the present patent application Goswami, Usha and Ganguly, Anutosh have already filed a patent application on a natural fluorescent dye extract from a marine invertebrate (U.S. patent application Ser. No. 09/820,654). This pertains to the crude extract from Holothuria scabra, which has the fluorescent qualities at three different wavelengths when, excited at different UV and visible ranges of the spectra of light. The invention also provides a process of the extraction, purification and characterization of this new dye, which is a partially purified natural dye from a sea cucumber. The utilities of the dye as an epifluorescent stain and non-radioactive fluorescent dye useful for labeling of molecular probes for in situ hybridization studies is described besides several other qualities of the dye as a drug. In this application, prior art details about the pigments, synthetic dyes and natural dyes from terrestrial plants and microbes have been described. (U.S. Pat. No. 4,452,822 published on Jun. 5, 1984; Inventors—Shrikhande, Anil J; U.S. Pat. No. 5,321,268 of Jun. 14, 1994 by Crosby David A and Ekstrom Philip A; U.S. Pat. No. 5,405,416 published on Apr. 11, 1995 authors Swinton; Robert J, U.S. Pat. No. 5,858,761 published on Jan. 12, 1999, inventors Tsubokura, et al. U.S. Pat. No. 5,902,749 of May 11, 1999 inventors Lichtwardt et al. U.S. Pat. No. 5,908,650 published on Jun. 1, 1999 inventors Lenoble, et al. U.S. Pat. No. 5,920,429 published on Jul., 6 1999 Burns et al U.S. Pat. No. 5,935,808 on Aug. 10, 1999 of Hirschberg, et al; U.S. Pat. No. 5,989,135 of Nov. 23, 1999 inventors Welch; David Emanuel; U.S. Pat. No. 6,055,936 issued on May 2, 2000; Collin; Peter Donald; U.S. Pat. No. 6,056,162 May 2, 2000; Leighley; Kenneth C.; U.S. Pat. No. 6,103,006 Aug. 15, 2000DiPietro; Thomas C.; U.S. Pat. No. 6,110,566 Aug. 29, 2000; White et al. U.S. Pat. No. 6,140,041 Oct. 31, 2000LaClair; James J. U.S. Pat. No. 6,165,384 Dec. 26, 2000 Cooper et al.; U.S. Pat. No. 6,180,154 Jan. 30, 2001 Wrolstad et al. EP0206718 published on Dec. 30, 1986 inventors Cramer Randall J; IE901379 of 30 Jan. 1991 Lee Linda G; Mize Patrick D; WO9010044 of July 7, 1990. Swinton; Robert J; AU704112 published on 7 Oct. 1997 inventors Burns David M; Pavelka Lee A; DE19755642 of 24 Jun. 1999 of Weimer Thomas D R.; WO9938919 28 Sep. 1999 Laclair James J; WO0058406 of 5 Oct. 2000 by Rosenblum Barnett B et al.; WO9938916 15 Aug. 2000 inventors DiPietro; Thomas C ; WO9920688 of 29 Aug. 2000 inventors Pavelka Lee et al.; WO9920688 of 29 Aug. 2000 inventors White et al.
The multiple uses of fluorescent dyes in the molecular biology research, in industrial applications and in life saving devices etc. are also described. Collin, P. D in his U.S. patents of Jun. 23, 1998, Mar. 2, 1999and Nov. 16, 1999 respective U.S. Pat. Nos. 5,770,205, 5,876,762 and 5,985,330 have described therapeutic properties of various body parts of sea cucumber. All these references also pertain to the present patent.
In the present patent applicants have adopted a different approach. In this invention, applicants have purified a chemical compound and a fluorescent dye, which is peculiar in its structure and in showing eight colored emissions for the first time. The chemical structure of the compound is disclosed for the first time from sea cucumber. Both the product and the source are new. The fluorophore part of the compound is phenolic and both the full compound and only the fluorophore have the properties of natural fluorescent dyes. Unlike the presently available dyes this compound is not synthetic derivative of any dyes currently sold in the market. It is a absolutely novel organosilicon Si—O—R type of compound, purified and disclosed for the first time in this invention. The process for the extraction, purification and characterization of this novel fluorescent polysaccharide from the body wall extract of the sea cucumber is also new and is disclosed for the first time. The fluorescent nature of any polysaccharide disclosed is also new and its uses as a natural multiple fluorescent dye for molecular probes is also new. The disclosure that the compound is of Si—O—R type and the core organic molecule is connected to a silicon matrix around it through the sulphate bonds is done here for the first time. The silicon matrix forms an integral part of the compound and the disclosure that it takes part in the metabolic activities of the sea cucumber is also new.
In yet another aspect, the invention provides the interested molecular probes and molecular chemical reagent companies with a new natural compound which can provide them with a fluorescent dye of high molecular and also of low molecular weight, depending upon the requirements.
Yet another aspect of the invention is that the compound is rich in sulphur and its various compositions show utility as insecticide, pesticide and a veterinary drug.
Unlike most other fluorescent synthetic dyes known, our dye does not need to be mixed with another dye for getting different fluorescence hues at different wavelengths in epifluorescence microscopy. After coming in contact with the bio-molecules it emits eight different colored fluorescence at eight different excitation wavelengths which can have multiple uses. It stains the cell membrane and emits blue, yellow and orangish red hues under different emission wavelengths of the fluorsescence microscope. Further, our dye is non-proteinaceous in nature and is highly stable at the room temperature for months and does not get contaminated by microbes. Its fluorescence quality doesn't get deteriorated at high and low temperatures unlike extracts of some algae and luminescent organisms. The dye does not show quick quenching effect when cell preparations are exposed to light under the microscope.
One important aspect of this fluorescent dye is its making compositions and kits for non-radioactive labeling of molecular probes and counterstaining. Its chemical structure is disclosed so the dye industry can synthesize on large scale. At different wavelength excitations it gives the effect equivalent to colors of wavelength spectrum of at least 123 flurochromes presently known in the market.
In yet another aspect, the dye when excited with X-rays of high energy show emissions in the longer ranges. It emits yellowish green fluorescence.
Yet another aspect is its use as a fluorochrome stain in epiflourescence microscopy which is reported here for the first time for any marine natural dye. The dye gives a counterstain effect of different cell components. This application provides a simple and quick method of checking cytogenetical preparations for multiple uses like molecular diagnostics using fluorescent in situ hybridization techniques, rapid diagnosis of bio-contamination in tissue cultures, industrial preparations, water quality check in laboratory and field conditions.
Yet another aspect of the dye is its use as a component of the non-radioactive labeling kits for advanced molecular biology applications and as a drug in medical and veterinary preparations.